U.S. patent application number 10/318124 was filed with the patent office on 2004-11-04 for capillary electrophoretic apparatus, sample plate and sample injection method.
Invention is credited to Fujiwake, Hideshi, Hayashizaki, Yoshihide, Hazama, Makoto, Nakamura, Shin.
Application Number | 20040217004 10/318124 |
Document ID | / |
Family ID | 27339198 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040217004 |
Kind Code |
A1 |
Hayashizaki, Yoshihide ; et
al. |
November 4, 2004 |
Capillary electrophoretic apparatus, sample plate and sample
injection method
Abstract
An electrode plate of a sample plate is set on the body of an
electrophoretic apparatus, while a plug is inserted into a
migration high voltage line connection hole and connected to a
high-tension distribution cable. Each well of a base plate is
inserted into a through hole of a well guide and further press-fit
and engaged into a cavity of an electrode plate, for fixing the
base plate to the electrode plate. Thereafter a sample is
introduced into each well of the base plate and an end of a
capillary column is dipped into each well for applying a migration
voltage and electrophoretically injecting the sample into the
capillary column.
Inventors: |
Hayashizaki, Yoshihide;
(Ibaraki, JP) ; Nakamura, Shin; (Kyoto, JP)
; Hazama, Makoto; (Kyoto, JP) ; Fujiwake,
Hideshi; (Kyoto, JP) |
Correspondence
Address: |
RADER FISHMAN & GRAUER PLLC
LION BUILDING
1233 20TH STREET N.W., SUITE 501
WASHINGTON
DC
20036
US
|
Family ID: |
27339198 |
Appl. No.: |
10/318124 |
Filed: |
December 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10318124 |
Dec 13, 2002 |
|
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09426760 |
Oct 26, 1999 |
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6517696 |
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Current U.S.
Class: |
204/455 ;
204/604; 422/400 |
Current CPC
Class: |
G01N 27/44743 20130101;
G01N 27/44782 20130101 |
Class at
Publication: |
204/601 ;
422/102; 204/604 |
International
Class: |
G01N 027/26; G01N
027/453 |
Claims
What is claimed is:
1. A sample plate comprising: a disposable insulating resin base
plate including one or a plurality of holes formed as wells having
open bottom portions projecting flush with each other; and an
electrode plate of conductive metal including cavities having
smaller sizes than the wells formed on positions corresponding to
two-dimensional arrangement of the wells formed by the base plate
for receiving press-fitted bottom portions of the wells thereby
fixing the base plate.
2. The sample plate in accordance with claim 1, wherein the base
plate is a strip-shaped one including a plurality of the wells
aligned with each other, and a plurality of the base plates are
aligned with each other thereby forming the two-dimensional
arrangement of the wells.
3. The sample plate in accordance with claim 1, further comprising:
a well guide, arranged between the base plate and the electrode
plate, having through holes on positions corresponding to the
cavities of the electrode plate.
4. A multi-capillary electrophoretic apparatus comprising: a
multi-capillary array migration part including a plurality of
capillary columns, having two-dimensionally arranged capillary
column ends downwardly directed on a sample injection side so that
a plurality of samples are injected one by one into the capillary
columns and simultaneously electrophoresed in all the capillary
columns; an optical measuring part irradiating the capillary
columns with light on the multi-capillary array migration part for
measuring absorbance by the samples or fluorescence from the
samples on irradiated portions; a sample plate comprising a
disposable insulating resin base plate, arranged under the
capillary column ends of the multi-capillary array migration part
on the sample injection side, including wells for storing samples,
two-dimensionally arranged in correspondence to arrangement of the
capillary column ends, having open bottom portions projecting flush
with each other and an electrode plate of conductive metal
including cavities having smaller sizes than the wells formed on
positions corresponding to two-dimensional arrangement of the wells
formed by arrangement of the base plate for receiving press-fitted
bottom portions of the wells thereby fixing the base plate; a
migration reservoir storing a migration buffer solution for
applying a voltage to all the capillary columns; and wherein the
sample plate and the migration reservoir are movable for switching
and bringing either one of these into contact with the capillary
column ends.
5. A sample injection method for injecting, in a capillary
electrophoretic apparatus employing a capillary column charged with
a gel of a separation carrier, a sample into the capillary column,
the sample injection method controlling high voltage application
for sample injection to gradually raise a voltage when starting
voltage application at the time of injecting the sample into the
capillary column with voltage application and to gradually lower
the voltage when finishing voltage application.
6. The sample injection method in accordance with claim 5, setting
an integrated value of the product of the applied voltage and time
equal to an integrated value of the product of the applied voltage
and time when keeping the sample injection voltage constant from
start to finish of voltage application.
7. A capillary electrophoretic apparatus comprising. a migration
part injecting a sample into an end of a capillary column charged
with a gel of a separation carrier and separating the injected
sample by electrophoresis; an optical measuring part irradiating
the capillary column with light in the migration part for measuring
absorbance by the sample or fluorescence from the sample on the
irradiated portion; and an applied voltage control part controlling
voltage application from a power source to the capillary column
when applying a voltage from the power source and injecting the
sample into the capillary column to gradually raise the voltage
when starting voltage application and to gradually lower the
voltage when finishing voltage application.
8. The capillary electrophoretic apparatus in accordance with claim
7, wherein the migration part is a multi-capillary array migration
part including a plurality of capillary columns so arranged that a
plurality of samples are injected one by one into the capillary
columns and simultaneously electrophoresed in all the capillary
columns.
9. A sample injection method for a capillary electrophoretic
apparatus comprising a migration part electrophoresing a sample
injected into an end of a capillary column charged with a gel of a
separation carrier and detection means detecting each component
separated in the capillary column on an appropriate position of the
capillary column, the sample injection method providing a
preliminary separation carrier for preliminary separation between
the sample and a capillary column sample injection end, injecting
the sample into the capillary column through the preliminary
separation carrier and removing the preliminary separation carrier
before starting the electrophoresis.
10. A capillary electrophoretic apparatus comprising: a migration
part electrophoresing a sample injected into an end of a capillary
column charged with a gel of a separation carrier; detection means
detecting each component separated in the capillary column on an
appropriate position of the capillary column; and a preliminary
separation part storing a preliminary separation carrier for
preliminary separation and the sample so that the preliminary
separation carrier is located between a sample injection end of the
capillary column and the sample.
11. The capillary electrophoretic apparatus in accordance with
claim 10, wherein the preliminary separation part comprises a fluid
gel as the preliminary separation carrier, and the sample is set in
the preliminary separation carrier in a state adsorbed by a filter
medium.
12. The capillary electrophoretic apparatus in accordance with
claim 10, wherein the preliminary separation part comprises a fluid
gel as the preliminary separation carrier and is so stratified that
an aqueous solution containing the sample forms an upper layer and
the preliminary separation carrier forms a lower layer, and the
sample injection end of the capillary column is inserted into the
preliminary separation carrier.
13. The capillary electrophoretic apparatus in accordance with
claim 10, wherein the migration part is a multi-capillary array
migration part including a plurality of capillary columns so
arranged that a plurality of samples are injected one by one into
the capillary columns and simultaneously electrophoresed in all the
capillary columns.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a capillary electrophoretic
apparatus comprising a capillary electrophoretic part
electrophoresing a sample injected into an end of a capillary
column and detection means detecting each component separated in
the capillary column at an appropriate position of the capillary
column, and inclusively, it relates to a method and an apparatus
characterized in sample injection into a capillary column.
[0003] 2. Description of the Prior Art
[0004] Besides having those that comprise a single capillary
column, capillary electrophoretic apparatuses also have a
multi-capillary electrophoretic apparatus comprising a
multi-capillary array migration part including a plurality of
capillary columns for injecting a plurality of samples one by one
into the capillary columns and simultaneously electrophoresing the
samples in all capillary columns and an optical measuring part
irradiating the capillary columns with light in the multi-capillary
array migration part for measuring absorbance by the samples on the
irradiated portion or fluorescence from the samples.
[0005] The capillary columns include those charged and not charged
with gels for migration.
[0006] A capillary electrophoretic apparatus is applied for the
separation of protein, and sequence determination for DNA. When
applied to sequence determination for DNA, the capillary
electrophoretic apparatus employs Sanger's reaction,
electrophoreses a DNA fragment sample prepared by labeling a primer
or a terminator with a fluorescent material and detects
fluorescence from the DNA fragment sample during migration for
determining the base sequence.
[0007] A DNA sequencer having high sensitivity, a high speed and
high throughput is necessary for sequence determination for DNA
having long base sequence such as a human genome. As one method, a
multi-capillary DNA sequencer prepared by arranging a plurality of
capillary columns in place of that employing a flat plate type slab
gel is proposed. With such a capillary column, a sample can not
only be readily handled or injected but also migrated at a high
speed and detected in high sensitivity, when compared to the slab
gel. If a high voltage is applied to the slab gel, a band is spread
or a temperature gradient is caused due to influence by Joulean
heat However, the capillary column hardly causes such problems and
can perform detection in high sensitivity with small band spreading
even if performing high-speed migration with application of a high
voltage.
[0008] In capillary electrophoresis, a sample is injected into a
capillary column under pressure or with application of a voltage. A
method of electrophoretically injecting a sample is widely employed
in general due to the simplicity of the apparatus structure,
easiness of operations and excellent controllability of
parameters.
[0009] In relation to the method of injecting the sample into the
capillary column, migrational separability in later migrational
separation must not be deteriorated and sample injection must be
simpler.
[0010] Whether the capillary column is charged with a gel or not,
one end of the capillary column must be dipped in a prepared
sample, the other end must be dipped in a buffer solution, and an
electrode such as a platinum wire must be dipped in the vicinity of
the end of the capillary column within the sample. In a structure
holding the electrode in the vicinity of the end of each capillary
column in electrophoretic sample injection, the electrode structure
for sample injection becomes complicated in the case of a
multi-capillary electrophoretic apparatus collectively arranging a
plurality of capillary columns in the form of an array for
simultaneously performing electrophoresis. After dipping the end of
the capillary column in a sample contained in a sample injection
container and performing injection by applying a voltage or the
like, the end of the capillary column must be transferred into a
reservoir containing a buffer solution for migration. Thus,
operations between sample injection and the start of migration are
troublesome, and it is convenient if the operations can be
automated.
[0011] In both of an electrophoretic apparatus comprising a single
capillary column and a multi-capillary electrophoretic apparatus,
migrational separability may be deteriorated in capillary
electrophoresis charged with a gel, depending on the sample
injection method. In the case of electrophoretically injecting a
sample, a prescribed high voltage is applied within several seconds
when starting voltage application and the high voltage is zeroed
within several seconds when finishing voltage application in a
conventional voltage application method, as shown in FIG. 4.
Referring to FIG. 4, the vertical axis shows the voltage (kV) and
the horizontal axis shows the time (second). In this example, a
voltage of 7.6 kV is applied for 30 seconds. However, If a high
voltage is applied to the capillary column, remarkable stress is
applied to a gel located on an end of the capillary column due to
heat generation or the like to hinder injection of the sample into
the capillary column, leading to such bad influence that the number
of bases separable in migrational separation is limited.
Furthermore, if the high voltage is abruptly applied, the gel may
be forced out from the end of the capillary column due to
electroosmosis flow. If the forced-out gel is damaged, the
migrational separation state is disadvantageously deteriorated.
[0012] In slab gel electrophoresis, a large molecule (referred to
as a macromolecule) such as template DNA having a size unreceivable
in a gel matrix does not enter the gel matrix part but remains on
an inlet for the gel matrix part. Even if force moving the
macromolecule by driving force of an electric field is applied to a
separation part in slab gel electrophoresis, the separation part
has sufficient resistance against this force due to its sufficient
volume to hardly become a problem. Also, in capillary
electrophoresis, a macromolecule does not enter a gel matrix part
but remains on an inlet for the gel matrix part (this phenomenon is
referred to as clogging). Capillary electrophoresis has the
following features: 1) the volume of a separation carrier is
extremely small, 2) the material for the separation carrier is not
a strong one such as an acrylamide gel but a viscous polymer
solution, and 3) electric field strength per unit length is large.
Therefore, force moving the macromolecule by driving force of an
electric field is large, and resistance against this force is
small. Thus, occurrence of clogging results in deterioration of a
separation pattern.
[0013] In DNA sequence determination, since a macromolecule in a
sample is template DNA, in order to solve the aforementioned
problem resulting from clogging, therefore, the template DNA must
be removed before injecting the sample into a capillary column. It
is possible to remove the template DNA by chemical pretreatment For
example, when preparing a DNA fragment sample by the Sanger's
method, an enzyme, antigen or antibody is bonded to a primer for
separating the DNA fragment sample from a template by enzyme
reaction or antigen-antibody reaction after preparing the sample.
However, such chemical pretreatment is troublesome and requires
much labor and time.
SUMMARY OF THE INVENTION
[0014] The first objective of the present invention is to provide a
multi-capillary electrophoretic apparatus simplifying sample
injection.
[0015] The second objective of the present invention is to provide,
in relation to a capillary electrophoretic apparatus, a sample
injection method and an apparatus suppressing deterioration of
migrational separability.
[0016] In order to attain the first objective of simplifying sample
injection of a multi-capillary electrophoretic apparatus, a
specific sample plate is employed in the present invention. The
sample plate comprises a disposable insulating resin base plate
including one or a plurality of holes formed as wells having open
bottom portions projecting flush with each other, and an electrode
plate of conductive metal including cavities having smaller sizes
than the wells formed on positions corresponding to two-dimensional
arrangements of the wells formed by arrangement of the base plate
for receiving press-fitted bottom portions of the wells thereby
fixing the base plate. The wells of the base plate are press-fitted
in and fixed to the cavities of the electrode plate to form spaces
for receiving samples, and the samples are introduced into the
wells respectively.
[0017] Such a base plate is disposable and hence can prevent
contamination. The base plate may comprise only a single well, "n"
number of wells aligned with each other, or wells two-dimensionally
arranged in "n" number of rows and "m" number of columns. The
material for forming the base plate is suitably prepared from
universal engineering plastic such as polypropylene or polyethylene
having chemical resistance and exceptional formability, in
consideration of the disposability.
[0018] When employing this sample plate, voltage application to a
plurality of wells becomes possible without employing a complicated
electrode wiring structure. Sample injection into capillary columns
of an electrophoretic apparatus employing a multi-capillary array
having a number of capillary columns especially becomes
simplified.
[0019] In a multi-capillary electrophoretic apparatus according to
the present invention employing this sample plate,
two-dimensionally arranged capillary column ends are downwardly set
on a sample injection side of a multi-capillary array migration
part, the aforementioned sample plate having two-dimensionally
arranged wells storing samples and a migration reservoir storing a
migration buffer solution for applying a voltage to all capillary
columns are arranged under the capillary column ends in
correspondence to the arrangement of the capillary column ends, and
the sample plate and the migration reservoir are movable for
switching and bringing either one of these into contact with the
capillary column ends.
[0020] In the multi-capillary electrophoretic apparatus, the
samples are introduced into the wells of the sample plate fixed to
the electrode plate in sample injection into the capillary columns,
and the sample plate is moved by the moving mechanism for dipping
ends of the respective capillary columns in the samples stored in
the wells. Thereafter a voltage is applied between the electrode
plate of the sample plate and other ends of the capillary columns
and the samples are electrophoretically injected into the capillary
columns. After sample injection, the sample plate is separated from
the ends of the capillary columns by the moving mechanism, and the
ends of the capillary columns are dipped into the buffer solution
contained in the migration reservoir. Thereafter a voltage is
applied between buffer solutions in reservoirs on both ends of the
capillary columns thereby performing migration. Thus, operations
from sample injection into the capillary columns to migration are
automatically performed.
[0021] In this way, the multi-capillary electrophoretic apparatus
employing the aforementioned sample plate according to the present
invention for sample injection and comprising the moving mechanism
switching and bringing either the sample plate or the migration
reservoir into contact with the capillary column ends can
automatically perform operations of sample injection into the
capillary columns and migration.
[0022] A method according to the present invention for attaining
the second objective of suppressing deterioration of migrational
separability is adapted to control voltage application at the time
of injecting a sample into a capillary column with voltage
application to gradually raise the voltage when starting voltage
application and gradually lower the voltage when finishing voltage
application. As an apparatus, an applied voltage control part
controlling voltage application in the aforementioned manner is
provided.
[0023] Thus, stress of a gel located on a capillary column end can
be reduced by suppressing abrupt heat generation and reduction.
Furthermore, generation of large electroosmosis flow can be
suppressed for preventing the gel from being forced out from the
capillary column end. Consequently, migrational separability can be
improved.
[0024] Another inventive method for attaining the second objective
is a method of providing a separation carrier for preliminary
separation between a sample and an end of a capillary column,
injecting the sample into the capillary column through the
preliminary separation carrier and removing the preliminary
separation carrier before starting electrophoresis.
[0025] A capillary electrophoretic apparatus according to the
present invention for carrying out the sample injection method
comprises a preliminary separation part storing a separation
carrier for preliminary separation and a sample so that the
preliminary separation carrier is located between an end of an
inserted capillary column and the sample.
[0026] In the sample injection method provided with the preliminary
separation part, a voltage is applied in preliminary separation so
that the sample starts moving toward the end of the capillary
column. At this time, a macromolecule contained in the sample also
starts moving toward the end of the capillary column and causes
clogging on the preliminary separation carrier. At the same time, a
target of analysis contained in the sample other than the
macromolecule passes through the preliminary separation carrier and
moves toward the end of the capillary column. When the target of
analysis enters the capillary column, the preliminary separation
carrier is removed and thereafter the target of analysis is
separated. Thus, the macromolecule unreceivable in the separation
carrier charged in the capillary column is removed in the stage of
preliminary separation. Due to this, no macromolecule exists in
separation analysis, and a separation pattern can be prevented from
deterioration resulting from clogging.
[0027] The foregoing and other objects, features, aspects and
advantages of the present invention will become more apparent from
the following detailed description of the present invention when
taken in conjunction with the accompanying drawing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1A is a schematic perspective view showing an exemplary
base plate used in a sample plate according to an embodiment of the
present invention;
[0029] FIG. 1B is a sectional view taken along the line Z-Z in FIG.
1A;
[0030] FIG. 2A is a schematic perspective view showing the sample
plate according to the embodiment;
[0031] FIG. 2B is a sectional view taken along the line Y-Y in FIG.
2A;
[0032] FIG. 3 is a schematic perspective view showing an embodiment
of a multi-capillary electrophoretic apparatus performing sample
injection with the sample plate of the embodiment;
[0033] FIG. 4 illustrates examples of a voltage applied in
conventional sample injection in a capillary electrophoretic
apparatus and time, with the vertical and horizontal axes showing
the voltage (kV) and the time (sec.) respectively;
[0034] FIG. 5 is a schematic perspective view showing a
multi-capillary electrophoretic apparatus according to another
embodiment of the present invention;
[0035] FIG. 6 illustrates examples of a voltage applied in sample
injection controlled by an applied voltage control part of the
embodiment and time, with the vertical and horizontal axes showing
the voltage (kV) and the time (sec.) respectively;
[0036] FIG. 7A conceptually illustrates an operation in sample
injection of an embodiment preventing deterioration of a separation
pattern resulting from clogging in relation to a single capillary
column;
[0037] FIG. 7B conceptually illustrates later migrational
separation; and
[0038] FIG. 8 is a schematic diagram showing another way of
introducing a sample in the embodiment
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] FIGS. 1A to 3 show an embodiment of the present invention
attaining the first objective of simplifying sample injection in a
multi-capillary electrophoretic apparatus.
[0040] FIG. 1A is a perspective view showing a base plate 101 in a
sample plate 100 according to the embodiment employed in the
present invention. FIG. 1B is a sectional view taken along the line
Z-Z in FIG. 1A. The base plate 101 is entirely formed by a thin
plastic material, and wells 102 of holes having open bottom
portions are arranged on its flat surface at regular intervals.
Each well 102 has a thin bottom portion coming into contact with an
electrode plate 104 and a thick upper portion receiving a capillary
column. The sizes of the open bottom portion and the open upper
portion are 0.7 mm to 2 mm and 4 mm to 4.5 mm respectively. Thus,
when the open bottom portion is closed with the electrode plate 104
as described later for storing a sample, the level can be raised
even if the amount of the sample is small while the capillary
column can be readily guided into the sample stored in the well
102.
[0041] FIG. 2A is a perspective view showing the sample plate 100
according to this embodiment FIG. 2B is a sectional view taken
along the line Y-Y in FIG. 2A.
[0042] The electrode plate 104 is made of a conductive material
such as stainless steel having high mechanical strength, and
vertically and horizontally arranged cavities 105 are formed on its
flat surface. A plurality of base plates 101 are arranged on the
surface of the electrode plate 104. The arrangement of the cavities
105 corresponds to two-dimensional arrangement of the wells 102 of
the base plates 101 arranged on the surface of the electrode plate
104. The size of each cavity 105 is slightly smaller than the outer
size of the bottom portion of each well 102, for fixing each base
plate 101 by press-fitting and engaging the bottom portion of each
well 102 into each cavity 105. A migration high voltage line
connection hole 107 is formed on the bottom portion of the
electrode plate 104 to be connected with a high voltage power
source through a plug 106 set on the body of the electrophoretic
apparatus. A well guide 103 guiding the wells 102 into the cavities
105 of the electrode plate 104 is fixed to the surface of the
electrode plate 104, and through holes are formed on positions
corresponding to the cavities 105. The well guide 103 may be made
of any material such as a conductor or an insulator. The base
plates 101, the well guide 103 and the electrode plate 104 form the
sample plate 100.
[0043] After use, the electrode plate 104 is detached from the base
plates 101. While the base plates 101 are discarded after single
use, the electrode plate 104 and the well guide 103 are repeatedly
used. The electrode plate 104 is washed for reuse. In particular,
the cavities 105 having portions coming into contact with samples
are necessarily washed for reuse.
[0044] The sample plate 100, which can be used not only in a gel
electrophoretic apparatus having capillary columns charged with
gels such as polyacrylamide gels, linear acrylamide gels or
polyethylene oxide gels as separation media but also in a capillary
zone electrophoretic apparatus performing free migration in
capillary columns charged with no gels, is applicable to any
electrophoretic apparatus so far as the same employs a plurality of
capillary columns.
[0045] FIG. 3 schematically shows an embodiment of a
multi-capillary electrophoretic apparatus performing sample
injection with the sample plate 100 and thereafter performing
migrational separation of samples.
[0046] A pair of reservoirs 110 and 120 store buffer solutions 112
and 112 respectively, and electrodes 130 and 132 are provided in
the buffer solutions 112 and 122 respectively. The sample plate 100
is that shown in FIG. 2A. Samples are introduced into the
respective wells 102 of the sample plate 100, and a high-tension
distribution cable is connected to the plug 106.
[0047] A high voltage switching part 136 switchably connects the
reservoir 110 and the sample plate 100 so that wiring can be
switched, and a high voltage power source 134 is connected between
the high voltage switching part 136 and the electrode 132 provided
in the other reservoir 120. The high voltage power source 134 is
connected with an applied voltage control part (not shown) control
applied voltages and times for sample injection and migration.
[0048] One capillary column ends 2a of a capillary array 2 are
inserted one by one in the respective wells 102 of the sample plate
100 in sample injection, while the reservoir 110 is switched to and
arranged on the position of the sample plate 100 after sample
injection, so that the one ends 2a of the capillary array 2 are
dipped in the buffer solution 112. The other ends 2b of the
capillary array 2 are dipped in the buffer solution 122 in the
other reservoir 120. The other ends 2b are provided with detected
portions 2c irradiated with measuring light or excitation light
from an optical measuring part 10 detecting the samples by
absorbance or fluorescence.
[0049] The capillary array 2 has two-dimensional arrangement
corresponding to the arrangement of the wells 102 of the sample
plate 100 on the side of the one ends 2a while capillary columns
are aligned with each other on the detected portions 2c so that the
arrangement surfaces of the capillary columns are perpendicularly
irradiated with the measuring light or the excitation light.
[0050] Each capillary column forming the capillary array 2 is made
of quartz glass or borosilicate glass (e.g., Pyrex) and has an
outer diameter of 200 to 300 .mu.m and an inner diameter of 75 to
100 .mu.m. The outer periphery of the capillary column is
preferably covered with a film of a non-fluorescent material such
as SiO.sub.2 generating no fluorescence by excitation light from
ultraviolet to near infrared regions or generating fluorescence to
an extent not hindering fluorescence measurement. In this case, the
film may not be removed on the detected portion 2c. If the
capillary column has a resin film generating fluorescence, the film
is removed on the detected portion 2c. A plurality of such
capillary columns are arranged in the capillary array 2.
[0051] The capillary columns are charged with polyacrylamide gels,
linear acrylamide gels or polyethylene oxide (PEO) gels as gels of
separation media. Samples containing four types of DNA fragments
labeled by four types of fluorescent materials selected from FAM,
JOE, TAMRA, ROX, R6G, R-110 and the like varying with end bases or
labeled in four types by at least two types of fluorescent
materials at different ratios are injected into the capillary
columns respectively and simultaneously electrophoresed.
[0052] The sample plate 100 and the reservoir 110 are so switched
and arranged by a moving mechanism (not shown in FIG. 3) that
either one thereof selectively comes into contact with the one ends
2a of the capillary array 2 as indicated by the broad arrow in FIG.
3.
[0053] The wells 102 not only store already treated samples, but
also can be so employed that sample injection can be performed by
treating samples by a PCR (polymerase chain reaction) method
through the wells 102 and thereafter inserting the one capillary
column ends 2a of the capillary array 2 into the samples stored in
the wells 102. The PCR method is a method of remarkably amplifying
only one target part of DNA. In the PCR method, a primer is added
to a sample of DNA, the temperature is raised to dissociate
double-stranded DNA to single chains. Then the temperature is
lowered to bond the primer with the DNA chains, the temperature is
slightly raised to synthesize DNA, and the temperature is further
raised to form single-stranded DNA. The operation of changing the
temperature upward and downward is repeated thereby
amplifying/synthesizing prescribed part of DNA in a large
quantity.
[0054] The electrode plate 104 of the sample plate 100 shown in
FIG. 2A is set on the body of the electrophoretic apparatus, while
the plug 106 is inserted into the migration high voltage line
connection hole 107 and connected to the high-tension distribution
cable. The wells 102 of the base plates 101 are inserted into the
through holes of the well guide 103 and further press fitted and
engaged into the cavities 105 of the base plates 101 for fixing the
base plates 101 to the electrode plate 104. Thereafter the samples
are introduced into the wells 102 of the base plates 101. If
necessary, centrifugal defoaming is lightly performed so that the
samples come into contact with the electrode plate 104.
[0055] In sample injection, the capillary column ends 2a on the
side of the one end of the capillary array 2 are dipped one by one
in the samples stored in the wells 102 of the sample plate 100
while the other ends of the capillary columns are collectively
dipped in the buffer solution 122 stored in the reservoir 120. A
high voltage is simultaneously applied to all wells 102 through the
electrode plate 104 for injecting the samples into the capillary
columns.
[0056] After sample injection, high voltage application is
temporarily stopped and the moving mechanism moves the sample plate
100 and the reservoir 110, thereby dipping the capillary column
ends on the side of the samples (the side of the one capillary
column ends 2a of the capillary array 2) into the buffer solution
112 stored in the reservoir 110. Thereafter, a high voltage is
applied between the reservoirs 110 and 120 for performing
electrophoretic separation. In order to connect a high voltage line
to the electrode plate 104, a plate spring or the like connected
with the high voltage power source 136 may be provided on a
position for fixing the sample plate 100 so that the plate spring
comes into contact with the electrode plate 104 when fixing the
sample plate 100. The moving mechanism may move the sample plate
100 and the reservoir 110 in a horizontal plane for vertically
moving the one capillary column ends 2a of the capillary array
2.
[0057] The number of the wells 102 of the sample plate 100 can be
arbitrarily set in response to the number of the capillary
columns.
[0058] FIG. 5 is a schematic perspective view showing an embodiment
of a capillary electrophoretic apparatus to which the present
invention for attaining the second objective of suppressing
deterioration of migrational separability is applied. While a
multi-capillary electrophoretic apparatus is shown, the present
invention is also applicable to a capillary electrophoretic
apparatus comprising a single capillary column.
[0059] A sample plate 100a, made of an insulating material,
comprises a flat surface and a connector portion 106a connected
with the same. A plurality of wells 102a are vertically and
horizontally arranged on the surface of the sample plate 100a at
regular intervals. The wells 102a are bottomed holes, and
individual electrode patterns reaching the connector portion 106a
from the bottoms through the base plate surface are formed on the
respective wells 102a. Samples are introduced into the respective
wells 102a of the sample plate 100a, and a high-tension
distribution cable is connected to the connector portion 106a.
[0060] The structure of this electrophoretic apparatus other than
the sample plate 100a is identical to that of the capillary
electrophoretic apparatus shown in FIG. 3, and hence redundant
description is omitted. An applied voltage control part 138
connected with a high voltage power source 134 for controlling
applied voltages and times for sample injection and migration is
implemented by a microcomputer or the like.
[0061] The one capillary column ends 2a of a capillary array 2 are
inserted one by one in the respective wells 102a of the sample
plate 100a in sample injection, while the sample plate 100a and a
reservoir 110 are switched after sample injection so that the one
capillary column ends 2a are dipped into a buffer solution 112. The
other ends 2b of the capillary array 2 are dipped into a buffer
solution 122 stored in another reservoir 120.
[0062] Operations of this embodiment shall now be described.
[0063] Samples are introduced into the wells 102a of the sample
plate 100a respectively. Thereafter the one capillary column ends
2a of the capillary array 2 are dipped one by one into the samples
stored in the wells 102a of the sample plate 100a, while the other
ends 2b of the capillary array 2 are collectively dipped in the
buffer solution 122 stored in the reservoir 120. The wells 102a are
connected with a high voltage power source 134 through a high
voltage switching part 136, the connector portion 106a and an
electrode pattern. A high voltage is applied between the wells 102a
and the buffer solution 122 by the high voltage power source 134,
for injecting the samples into capillary columns. At this time, the
applied voltage control part 138 controls the applied voltage and
the time.
[0064] FIG. 6 illustrates examples of the voltage applied in sample
injection and the time controlled by the applied voltage control
part 138. The horizontal and vertical axes show the voltage (kV)
and the time (sec.) respectively. The applied voltage control part
138 controls the high voltage power source 134, for gradually
raising the voltage to reach 7.6 kV after a lapse of 15 seconds
from start of sample injection. Thereafter the voltage of 7.6 kV is
applied for 15 seconds. Then, the voltage is lowered from 7.6 kV to
0 V over 15 seconds, and voltage application in sample injection is
terminated. An integrated value (area of the slant line part) of
the product of the applied voltage and the time is set to be equal
to an integrated value (area of the slant line part) of the product
of the applied voltage and the time shown in FIG. 4. The applied
voltage and the time vary with the types of the samples or the type
of the apparatus, and hence the integrated value thereof varies
with the types of the samples or the type of the apparatus.
[0065] Abrupt heat generation and reduction in gels in the
capillary columns can be suppressed by controlling the applied
voltage and the time in the aforementioned manner, for relaxing
stress on the gels located on capillary column ends. Furthermore,
it is also possible to suppress occurrence of large electroosmosis
flow and prevent the gels from being forced out from the capillary
column ends. In addition, it is also possible to suppress overshoot
of a current resulting from abrupt application of the high
voltage.
[0066] After sample injection, the moving mechanism moves the
sample plate 10a and the reservoir 110 thereby dipping the one ends
2a of the capillary array 2 on the side of the samples into the
buffer solution 112 stored in the reservoir 110. Thereafter a high
voltage is applied between the reservoirs 110 and 120 for
performing electrophoretic separation. A power supply voltage for
migration is for example, 30 kV, and a current capacity is 10 to 30
mA. Migrational separability, which is about 100 bases in general,
can be improved to 200 to 300 bases by injecting the samples into
the capillary columns in the aforementioned manner.
[0067] The reservoir 110 may also have a plurality of wells storing
buffer solutions similar to the sample plate 100a so that
independent voltages can be applied to the respective wells by
providing individual electrode patterns on the respective wells for
simultaneously performing electrophoresis under different
conditions.
[0068] FIG. 7A conceptually illustrates an operation in sample
injection of an embodiment for preventing deterioration of a
separation pattern resulting from clogging in relation to a single
capillary column. A capillary electrophoretic apparatus to which
this embodiment is applied is not restricted to that shown in FIG.
5 but the multi-capillary electrophoretic apparatus shown in FIG. 3
or still another capillary electrophoretic apparatus is also
employable. The following description is made with reference to the
multi-capillary electrophoretic apparatus shown in FIG. 5 as an
example.
[0069] The sample plate 100a defines a preliminary separation part
serving also as a sample injection part. A fluid gel (separation
carrier for preliminary separation) 203 and a sample 205 adsorbed
by filter paper are contained in each well 102a of the preliminary
separation part 100a so that the gel 203 is interposed between a
capillary column 2 and the sample 205. A wiring pattern is formed
on the preliminary separation part 100a and an electrode 107a is
inserted in each well 102a, while the electrode 107a is connected
to a high-tension distribution cable through the connector portion
106a. Although the electrode 107a is shown as a wire, it is formed
as a thin-Film pattern extending from the surface of the
preliminary separation part 10a to the well 102a.
[0070] The capillary column 2 forming a capillary array is charged
with a fluid gel (separation carrier) 201 identical to the gel 203.
The one end 2a of the capillary column 2 is inserted in each well
102a of the preliminary separation part 100a in sample injection
and then dipped into the buffer solution 112 stored in the
reservoir 110 after sample injection. The other end 2b of the
capillary column 2 is dipped into the buffer solution 122 stored in
the other reservoir 120, and irradiated with measuring light or
excitation light from the optical measuring part 10 detecting the
sample 205 by absorbance or fluorescence.
[0071] The gels 201 and 203 can be prepared by fluid gels
consisting of 2% HEC (hydroxyethyl cellulose: product by
Polyscience Co., Ltd. (U.S.A.), 7M urea or .times.1 TBE (tris
borate buffer). In this embodiment, the gels 201 and 203 are the
same, but they do not have to be.
[0072] The operations of this embodiment shall now be described
with reference to FIGS. 5, 7A and 7B.
[0073] In sample injection, the one ends 2a of the capillary
columns are dipped one by one into the gels 203 stored in the wells
102a, while the other ends 2b of the capillary columns are
collectively dipped into the buffer solution 122 stored in the
reservoir 120. The high voltage switching part 136 is connected to
the preliminary separation part 100a for applying a high voltage
between each well 102a and the reservoir 120 by the high voltage
power source 134 for electrophoresis (FIG. 7A).
[0074] A target of analysis such as DNA contained in each sample
205 starts moving toward each capillary column 2. At this time, a
macromolecule such as template DNA simultaneously starts to move,
but cannot enter a matrix of the gel 203 and causes clogging, and
remains on a boundary surface between the gel 203 and the sample
205. At the same time, the target of analysis passes through the
gel 203 and moves toward the gel 201 in the capillary column 2, and
the sample 205 is injected.
[0075] After injection of the object of analysis, high voltage
application is temporarily stopped and the moving mechanism moves
the preliminary separation part 100a and the reservoir 110 thereby
dipping the one end 2a of the capillary column 2 on the side of the
sample 205 into the buffer solution 112 stored in the reservoir
110. Thereafter a high voltage is applied between the reservoirs
110 and 120 for performing electrophoretic separation (FIG. 7B). At
this time, the injected sample 205 does not contain macromolecule,
and hence deterioration of a separation pattern does not occur by
clogging. The voltage for sample injection into the capillary
column 2 and power supply voltage for migration are for example, 30
kV, and current capacity is 10 to 30 mA.
[0076] FIG. 8 is a schematic diagram showing another sample
injection method in this embodiment A sample is generally prepared
as an aqueous solution. Therefore, an aqueous solution 109
containing a sample and a gel 203 are stratified in a well 102a.
The gel 203 has higher density than the aqueous solution 109, and
hence the aqueous solution 109 can be stratified as an upper
layer.
[0077] One capillary column ends 2a of a capillary array 2 are
inserted into the gel 203 and a voltage for sample injection is
applied, so that a macromolecule in the sample contained in the
aqueous solution 109 causes clogging and remains on the interface
between the aqueous solution 109 and the gel 203 while the object
of analysis passes through the gel 203 and is injected into the
capillary column.
[0078] The number of wells of a preliminary separation part 100a
can be arbitrarily set in response to the number of capillary
columns.
[0079] It is also preferable to enable automatic performance of the
aforementioned operations.
[0080] The sample injection methods described with reference to
FIGS. 6 to 8 are also applicable to the capillary electrophoretic
apparatus shown in FIG. 3 employing the sample plate shown in FIG.
2A, if the capillary columns are charged with gels.
[0081] Although the present invention has been described and
illustrated in detail, it is clearly understood that the same is by
way of illustration and example only and is not to be taken by way
of limitation as the spirit and scope of the present invention are
limited only by the terms of the appended claims.
* * * * *